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ARS Home » Midwest Area » Peoria, Illinois » National Center for Agricultural Utilization Research » Bioenergy Research » Research » Research Project #436784

Research Project: Biochemical Technologies to Enable the Commercial Production of Biofuels from Lignocellulosic Biomass

Location: Bioenergy Research

2020 Annual Report

Objective 1. Develop technologies that enable the commercial production of marketable lipid-based advanced biofuels from lignocellulosic biomass hydrolyzates. Objective 2. In collaboration with industrial biorefiners, develop technologies that enable widespread commercial production of cellulosic ethanol from lignocellulosic biomass. Objective 3. Develop technologies that serve as co-products for lignocelluloses based refineries and as antibiotic alternatives for use in agriculture and animal production.

Goal 1. Develop oleaginous yeast and associated processes for converting hydrolyzates of lignocellulosic biomass to lipids for biodiesel and valuable coproducts for other uses. Goal 2. Apply novel patented stress-tolerant yeast strains under commercial conditions to convert hydrolyzates of lignocellulosic biomass to ethanol. Goal 3. Develop protective soil bacteria and yeast engineered with bacterial AMP genes for cultivation on biorefinery substrates to supply new antimicrobials for animals and agriculture.

Progress Report
This is a bridging project that was initiated on 7/25/2019, replacing 5010-41000-162-00D and to be replaced by 5010-41000-189-00D, “New Bioproducts for Advanced Biorefineries” and 5010-41000-190-00D, “Technologies to Improve Conversion of Biomass-Derived Sugars to Bioproducts.” It reports current progress for the same important objectives and incorporates the final report for the original project plan. Objective 1: Around 1.3 billion tons of plant biomass (lignocellulose) can be harvested each year in the United States. Assuming conservative cropping and yield estimates, this biomass could theoretically be converted to approximately 30 billion gallons of biodiesel/year (as much as 62% of current U.S. diesel consumption) using “oily” yeasts. Over the project lifetime, a major goal was to identify elite oily yeast strains able to grow on and use diverse concentrated sugars found in severely inhibitory lignocellulosic hydrolyzates that are typically produced because of low cost. ARS scientists in Peoria, IL, screened oily strains from the ARS Culture Collection (NRRL) on two commercially promising hydrolyzates of plant biomass (acid-pretreated switchgrass and ammonia fiber explosion-pretreated corn stover) to identify four elite robust novel yeast strains (Lipomyces tetrasporus, Lipomyces kononenkoae, Rhodosporidium toruloides, and Saitoella coloradoensis) able to produce high lipid titers with fatty acid profiles favorable for biodiesel. An advanced two-stage process with low carbon:nitrogen ( C:N) applied first to support growth followed by very high C:N second allowed top strains to consume diverse sugars and inhibitory acetic acid to produce 30 g/L lipids, an economically harvestable concentration, even under acidic pH conditions preferred by industry. This year in bioprocess development studies, a novel ammonia based thermal mechanical process was developed that reduced ammonia loading over three-fold and is expected to provide concentrated sugars with fewer inhibitors and lower nitrogen content to favor production of high lipid titers in oily yeasts. In FY2019-2020 adaptive evolution was designed to enrich and isolate variants of the four elite strains with shortened lag phase, more rapid growth and higher lipid accumulation in high solids dilute acid switchgrass hydrolyzates. To further broaden genetic diversity ARS scientists developed a novel method to select for native oily yeast from natural environments. These approaches made new superior yeast strains available to the public. New strains are expected to advance the economic feasibility of high-quality biodiesel and jet fuels from renewable herbaceous and woody biomass, reducing our dependence on foreign oil while supporting rural economy and the environment. Objective 2: Bioprocess and genetic engineering strategies were developed to support a yeast-based bioconversion that could substantially lower the selling cost of cellulosic ethanol and enhance rural economic development. ARS scientists in Peoria, IL, designed a strain assessment based on conditions desired for industrial applications in order to identify best practical strains for improved efficiency and cost of cellulosic ethanol production. Assessing utility under standard industrial conditions, this strain deposited as NRRL Y-50464 was confirmed to produce over 40 g/L ethanol from pure cellulose within 72 hours at a conversion rate of 0.04 g/L/h, applying conventional cellulase without the need to add supplementary beta-glucosidase enzyme. This new strain and process may allow enzyme cost reduction and consolidated process efficiencies providing an estimated savings of approximately $0.35/gal in the selling price of ethanol. Although strain Y-50464 is able to produce beta-glucosidase to aid saccharification of cellulose during ethanol production from corn stover, the conventional bioreactor designed for traditional low solids liquid fermentation is not suitable for the high solids content of typical simultaneous saccharification fermentation (SSF) operation. In this research, ARS scientists collaborated with Chinese scientists to design a new bioreactor with a helical stirring apparatus to provide sufficient mixing power during saccharification of the cellulose and ethanol production. It significantly reduced the cost of enzyme needed for SSF, and the ethanol yield was near that needed for industrial production. In related research (Objective 2), scientists continued to identify key genes, renovated pathways and mechanisms underlying the inhibitor tolerance of a patented industrial yeast Saccharomyces cerevisiae NRRL Y-50049, which was obtained by adaptive evolution when challenged with furanaldehydes: --five new genes from the native pentose-using yeast, Scheffersomyces stipitis, as a source of resistance against the metabolic inhibitor furfural; --inhibitor sensor genes, responding to presence of furanaldehydes, useful to engineering enhanced protective mechanisms for cell survival and hydrolysate detoxification mechanisms facilitating bioconversion; --a key protein found to serve as the basis for a fine-tuned mechanism of in situ inhibitor detoxification by the evolved inhibitor tolerant industrial yeast; and --ribosomal proteins critical for the yeast adaptation to toxic chemicals. Additionally, in FY20 an ARS scientist in Peoria, IL, discovered 79 transposable elements (TE) in the evolved tolerant patented strain Saccharomyces cerevisiae NRRL Y-50049 which showed significantly increased expressions in response to furanaldehyde challenge compared to the parent. TEs are believed to play significant roles in gene expression and genome evolution in many species, but this is the first report of TEs showing potential impact on yeast tolerance of toxic chemicals. Traditional industrial Saccharomyces yeast strains do not ferment pentose sugars, which comprise about one-third of available sugars from biomass. Efficient xylose conversion is critical to producing ethanol economically from biomass, and although S. cerevisiae strains have been engineered to use xylose, they have been deficient in robustness when cultivated on this sugar in inhibitory hydrolyzates. ARS scientists from Peoria, IL, discovered a coordinated gene expression system key to xylose utilization which allowed an engineered industrial yeast to better utilize xylose for increased ethanol conversion and biosynthesis. The scientists found that expressions of one set of genes led to a signature expression of 10 additional genes critical for the acquired xylose to ethanol conversion. This is the first report of coordinated pathway expression in an industrial yeast engineered to successfully use xylose and is a notable advance. These discoveries build a genetic basis for mechanisms of inhibitor tolerance and enhanced pentose utilization and provide insight into strategies of strain improvement. Robust industrial yeast strains are vital to low cost fuels and chemicals from renewable plant biomass and the advancement of national energy independence, strong rural economy, and preservation of the environment. Objective 3: Fusarium dry rot, incited by Fusarium sambucinum, causes greater losses in storage and transit of both seed and commercial potatoes than any other postharvest disease. Chemical options for control of the causative fungus are limited since 80% of pathogenic strains are resistant to thiabendazole, the only chemical registered for use on food-grade potatoes. There is growing pressure to develop non-azole alternatives although current chemical fungicides being considered to fill the thiabendazole void also contain azoles. As a microbial alternative, three Pseudomonas strains antagonistic to F. sambucinum were discovered in previous work, and a triculture product capable of superior efficacy in protecting potatoes against dry rot, late blight, and pink rot, and sprouting was developed by ARS scientists in Peoria, IL. During the current project, desiccation tolerant derivatives of the strains were developed by targeted adaptive evolution using a growth-drying-storage cycle to enrich for desired strains with an improved shelf life after air drying. The new desiccation tolerant bacteria were next shown to be able to grow well individually and as a triculture in switchgrass hydrolysate that was appropriately buffered to optimize pH near neutral. Experiments in progress indicate successful survival in dry storage at least several weeks. Following rehydration optimization, the dried triculture was efficacious against Fusarium dry rot and Pythium leak in bioassays conducted in collaboration with ARS and University of Idaho scientists. In related research, ARS scientists in Peoria, IL, discovered a new osmoprotective sugar (isomelezitose) that was found to be more protective of Pseudomonas strain viability during drying and storage than other sugars of similar structure currently used in industry. In prior work, ARS scientists developed a bioprocess to potentially make this sugar at relatively low cost, which would allow it to be economical for agricultural use. Production of a broad-spectrum antifungal agent on low cost renewable substrates and improved dry storage formulations of the biocontrol product are expected to lower costs and expedite application by growers. This new technology benefits agriculture by providing an antifungal microbial alternative to azole chemicals no longer effective due to pathogen resistance and by serving as a potential co-product of a renewable lignocellulose biorefinery with the effect of boosting economic feasibility.

1. Multigene profile of microbial inhibitor tolerance. One barrier to conversion of renewable plant biomass to biofuels and chemicals is the occurrence of microbial inhibitors during various steps of this process. An inhibitor tolerance phenotype is commonly reported based on expression of a single gene. However, this practice is insufficient to fully describe the responsible genetic code and gene interactions. Using whole genome studies, an ARS scientist in Peoria, Illinois, found at least four major gene groups comprised of over 100 genes coding metabolic routes and regulators responsible for the inhibitor tolerance phenotype of an ARS patented evolved industrial yeast Saccharomyces cerevisiae NRRL Y-50049. This research supports a new concept to define a true tolerance phenotype in terms of key pathway-based gene groups, and it established the first true tolerance phenotype as a foundation for in-depth study of underlying mechanisms of yeast tolerance. Robust industrial yeast strains are vital to low cost fuels and chemicals from renewable plant biomass and the advancement of national energy independence, strong rural economy, and preservation of the environment.

2. New biorefinery compatible oily yeast strain platform for synthetic biology. Over one billion tons of cellulosic biomass can be available throughout the United States without impinging on corn and other row crop production. ARS researchers in Peoria, Illinois, identified a promising new yeast named Candida phangngensis that would be suitable for commercial development because it is closely related to pre-existing commercial yeast strains known to be amenable to development using synthetic biology approaches and generally regarded as safe (GRAS). The yeast strain produces plant-like oils from cellulosic sugars. It was further demonstrated that the yeast could be transformed and exogeneous genes expressed, which improved its ability to grow on biomass sugars and to produce more oil. New strains are expected to advance the economic feasibility of high-quality biodiesel and jet fuels from renewable herbaceous and woody biomass, reducing our dependence on foreign oil while supporting rural economy and conserving the environment.

3. Improved biocontrol agent production and delivery technologies for potato disease control. Fusarium dry rot is a potato disease without effective control options since 80% of the responsible pathogens are resistant to chemical fungicides once used. ARS scientists in Peoria, Illinois, developed a rehydration process to allow overnight reactivation of a desiccation tolerant biocontrol agent (BCA). The rehydration procedure was compatible with promising new chemical fungicides (StadiumTM and Graduate A+TM ) and was effective with the reactivation of dried BCA formulations grown on either synthetic medium or on a potentially lower cost medium with sugars from hydrolyzed switchgrass, a herbaceous lignocellulosic feedstock being developed by ARS researchers in Lincoln, Nebraska, for biorefining. BCA treatments assessed at the ARS location in Peoria, Illinois, and small pilot trials achieved significant disease reduction on the Clearwater Russet potatoes. While mixtures of the BCA with fungicide were compatible with retaining BCA active viable cells, they significantly benefited reduced fungicide applications in low volume sprays. These results support use of triculture BCA alone or in combination with chemicals, which is of significance in broadening the number of modes of action available to growers to accomplish effective disease suppression and to reduce opportunity for resistance buildup by pathogens. The data also support this BCA as a potential higher value coproduct that could enhance the product portfolio breadth and value, and contribute to the economy of biorefinery operation and rural agriculture.

Review Publications
Liu, Z., Huang, X., Zhou, Q., Xu, J. 2019. Protein expression analysis revealed a fine-tuned mechanism of in situ detoxification pathway for the tolerant industrial yeast Saccharomyces cerevisiae. Applied Microbiology and Biotechnology. 103:5781–5796.
Liu, Z.L., Ma, M. 2020. Pathway-based signature transcriptional profiles as tolerant phenotypes for the adapted industrial yeast Saccharomyces cerevisiae resistant to furfural and HMF. Applied Microbiology and Biotechnology. 104:3473–3492.
Cheng, M., Sun, L., Jin, Y., Dien, B.S., Singh, V. 2020. Production of xylose enriched hydrolysate from bioenergy sorghum and its conversion to ß-carotene using an engineered Saccharomyces cerevisiae. Bioresource Technology. 308. Article 123275.
Dien, B.S., Anderson, W.F., Cheng, M., Knoll, J.E., Lamb, M., O Bryan, P.J., Singh, V., Sorensen, R.B., Strickland, T.C., Slininger, P.J. 2020. Field productivities of Napier grass for production of sugars and ethanol. ACS Sustainable Chemistry & Engineering. 8(4):2052-2060.
Cheng, M., Wang, Z., Dien, B.S., Slininger, P.J., Singh, V. 2019. Economic analysis of cellulosic ethanol production from sugarcane bagasse using a sequential deacetylation, hot water and disk-refining pretreatment. Processes. 7(10): 1-15.
Chen, M.H., Dien, B.S., Lee, D.K., Singh, V. 2019. Sugar production from bioenergy sorghum by using pilot scale continuous hydrothermal pretreatment combined with disk refining. Bioresource Technology. 289:121663.
Jordan, J.H., Easson, M.W., Dien, B., Thompson, S., Condon, B.D. 2019. Extraction and characterization of nanocellulose crystals from cotton gin motes and cotton gin waste. Cellulose. 26(10):5959-5979.
Wang, Z., Dien, B.S., Rausch, K.D., Tumbleson, M.E., Singh, V. 2019. Improving ethanol yields with deacetylated and two-stage pretreated corn stover and sugarcane bagasse by blending commercial xylose-fermenting and wild type Saccharomyces yeast. Bioresource Technology. 282:103-109.
You, J., Johnston, D., Dien, B.S., Singh, V., Engeseth, N.J., Tumbleson, M., Rausch, K.D. 2020. Effects of nitrogenous substances on heat transfer fouling using model thin stillage fluids. Food and Bioproducts Processing. 119:125-132.
Nichols, N.N., Mertens, J.A., Dien, B.S., Hector, R.E., Frazer, S.E. 2019. Recycle of fermentation process water through mitigation of inhibitors in dilute-acid corn stover hydrolysate. Bioresource Technology. 9:100349.
Kumar, A.K., Parikh, B., Liu, Z., Cotta, M.A. 2018. Application of natural deep eutectic solvents in biomass pretreatment, enzymatic saccharification and cellulosic ethanol production. Materialstoday: Proceedings. 5(11):23057-23063 pt. 2.